This invention relates to a novel enzyme, namely a novel enzyme which acts upon side chain amido groups in protein and thereby releases side chain carboxyl groups and ammonia, and to a production method thereof. More particularly, it relates to a method for the production of an enzyme having a property to deamidate amido groups in protein, which comprises culturing a bacterial strain capable of producing an enzyme having a property to deamidate amido groups in protein, that belongs to Cytophagales or Actinomycetes, more particularly to the genus Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium or Myroides, in a medium, thereby allowing the strain to produce said enzyme, and subsequently collecting said enzyme from the culture mixture. This invention also relates to a method for the modification of protein, which uses a novel enzyme having a property to directly act upon amido groups in protein. It also relates to an enzyme which has a property to deamidate amido groups in protein, to a gene which encodes said enzyme, to a vector which contains said gene, to a transformant transformed with said vector, and to a method for the production of an enzyme having a property to deamidate amido groups in protein, which comprises culturing said transformant in a medium, thereby allowing the transformant to produce said enzyme, and subsequently collecting said enzyme from the culture mixture.
Glutaminase/asparaginase are enzymes which hydrolyze glutamine/asparagine to convert them into glutamic acid/aspartic acid and ammonia, and it is well known that these enzymes are obtained from animals, plants and microorganisms. However, these enzymes specifically act on free glutamine/asparagine and cannot deamidate glutamine/asparagine in peptides or polypeptides.
Also, transglutaminase is known as an enzyme which acts upon amido groups existing in a peptide state. Transglutaminase catalizes the reaction of introducing an amine compound into protein by covalent bonding or the reaction of cross-linking the glutamine residue and lysin residue of protein via xcex5-(xcex3-glutamyl)lysine-isopeptide bonding, in which the amido group of peptide bonded glutamine as an acyl donor and the amino group of the primary amine as an acyl acceptor. It is known that, when amine or lysin does not exits in the reaction system or blocked, water acts as an acyl acceptor and the glutamine residue in paptide is deamidated to become glutamic acid residue. As described above, transglutaminase is basically an acyl group transferase. Accordingly, when allowed to act on a usual protein, this enzyme causes cross-linking of protein and does not deamidate the protein. Accordingly, transglutaminase is different from the enzyme of the present invention.
In addition, Peptideglutaminase I and Peptideglutaminae II produced by Bacillus circulans are known as an enzyme which performs deamidation by acting upon glutamine bonded in peptide. It is known that the former acts on the glutamine residue existing at the C terminal of peptide and the latter acts on the glutamine residue existing in the peptide. However, these enzymes hardly acts upon a high molecular weight protein and acts only upon a low molecular weight peptide [M. Kikuchi, H. Hayashida, E. Nakano and K. Sakaguchi, Biochemistry, vol. 10, 1222-1229 (1971)].
Also, plural studies have been made to attempt to allow these enzyme (Peptideglutaminase I and II) to act upon a high molecular weight protein rather than a low molecular weight peptide. As a result, it has been revealed that these enzymes do not substantially act on a high molecular weight protein but act only on a protein hydrolysate peptide. Gill et al. reported that each of Peptideglutaminase I and II does not act on milk casein and whey protein both in native form and denatured form. They also reported that, as a result of studies on activities on protein hydrolysate, only Peptideglutaminase II acted only on peptide having a molecular weight of 5000 or less (B. P. Gill, A. J. O""Shaughnessey, P. Henderson and D. R. Headon, Ir. J. Food Sci. Technol., vol. 9, 33-41 (1985)). Similar studies were carried out by Hamada et al. using soy bean protein and the result was consistent with the result by Gill et al. That is, it was reported that these enzymes showed deamidation percentage of 24.4% to 47.7% on soy bean peptide (Peptone), but did not substantially act on soy bean protein (0.4% to 0.8%) (J. S. Hamada, F. F. Shih, A. W. Frank and W. E. Marshall, J. Food Science, vol. 53, no. 2, 671-672 (1988)).
There is an report suggesting existence of an enzyme originating from plant seed, which catalyzes deamidation of protein (cf. I. A. Vaintraub, L. V. Kotova, R. Shara, FEBS Letter, Vol. 302, 169-171 (1992)). Although this report observed ammonia release from protein using a partially purified enzyme sample, it is clear that this report does not prove existence of an enzyme of the present invention from the following reasons. In this report, a partially purified enzyme sample was used, inexistence of protease activity was not confirmed, and no change in molecular weight of substrate protein after the reaction was not confirmed. Accordingly, this report does not exclude the possibility that plural enzymes (not one enzyme) such as protease, peptidase, etc. acted on protein to release glutamine/asparagine as free amino acids and ammonia was released by glutaminase/asparaginase which deamidate these free amino acids. Similarly, there is a possibility that glutamine-containing low molecular weight peptide produced in a similar way is deamidated by peptideglutaminase-like enzyme. In addition, there is a possibility that deamidation occurred as a side-reaction by protease. In particular, it should be noted that this report clearly describes that the partially purified preparation had glutaminase activity which acted on free glutamine to release ammonia.
Accordingly, there is no report until now which confirmed existence of an enzyme which can catalyzes deamidation of on high molecular weight protein by purification of the enzyme as a single protein and isolation and expression of the gene encoding the same.
In general, when carboxyl groups are formed by deamidation of glutamine and asparagine residues in protein, negative charge of the protein increases and, as the results, its isoelectric point decreases and its hydration ability increases. It also causes reduction of mutual reaction between protein molecules, namely, reduction of association ability, due to the increment of electrostatic repulsion. Solubility and water dispersibility of protein sharply increases by these changes. Also, the increment of negative charge of protein results in the change of the higher-order structure of the protein caused by loosening of its folding, thus exposing the hydrophobic region buried in the protein molecule to the molecular surface. In consequence, a deamidated protein has amphipathic property and becomes an ideal surface active agent, so that emulsification ability, emulsification stability, foamability and foam stability of the protein are sharply improved.
Thus, deamidation of a protein results in the improvement of its various functional characteristics, so that the use of the protein increases sharply (for example, see Molecular Approaches to Improving Food Quality and Safety, D. Chatnagar and T. E. Cleveland, eds., Van Nostrand Reinhold, New York, 1992, p. 37).
Accordingly, a large number of methods for the deamidation of protein have been studied and proposed. An example of chemical deamidation of protein is a method in which protein is treated with a mild acid or a mild alkali under high temperature condition. In general, amido groups of glutamine and asparagine residues in protein are hydrolyzed by an acid or a base. However, this reaction is non-specific and accompanies cutting of peptide bond under a strong acid or alkali condition. It also accompanies denaturation of protein to spoil functionality of the protein.
Accordingly, various means have been devised with the aim of limiting these undesired reactions, and a mild acid treatment (for example, see J. W. Finley, J. Food Sci., 40, 1283, 1975; C. W. Wu, S. Nakai and W. D. Powie, J. Agric. Food Chem., 24, 504, 1976) and a mild alkali treatment (for example, see A. Dilollo, I. Alli, C. Biloarders and N. Barthakur, J. Agric. Food Chem., 41, 24, 1993) have been proposed. In addition, the use of sodium dodecyl sulfate as an acid (F. F. Shih and A. Kalmar, J. Agric. Food Chem., 35, 672, 1987) or cation exchange resin as a catalyst (F. F. Shih, J. Food Sci., 52, 1529, 1987) and a high temperature treatment under a low moisture condition (J. Zhang, T. C. Lee and C. T. Ho, J. Agric. Food Chem., 41, 1840, 1993) have also been attempted.
However, all of these methods have a difficulty in completely restricting cutting of peptide bond. The cutting of peptide bond is not desirable, because it inhibits functional improvement of protein expected by its deamidation and also causes generation of bitterness. Also, the alkali treatment method is efficient in comparison with the acid treatment method, but it has disadvantages in that it causes racemization of amino acids and formation of lysinoalanine which has a possibility of exerting toxicity.
On the other hand, some enzymatic deamidation methods have also been attempted with the aim of resolving the aforementioned problems of the chemical methods. Namely, a protease treatment method under a high pH (pH 10) condition (A. Kato, A. Tanaka, N. Matsudomi and K. Kobayashi, J. Agric. Food Chem., 35, 224, 1987), a transglutaminase method (M. Motoki, K. Seguro, A. Nio and. K. Takinami, Agric. Biol. Chem., 50, 3025, 1986) and a peptideglutaminase method (J. S. Hamada and W. E. Marshall, J. Food Sci., 54, 598, 1989) have been proposed, but each of these methods has disadvantages.
Firstly, the protease method cannot avoid cutting of peptide bond as its original reaction. As described in the foregoing, cutting of peptide bond is nor desirable.
In the case of the transglutaminase method, it is necessary to chemically protect xcex5-amino group of lysine residue in advance, in order to prevent cross-linking reaction caused by the formation of isopeptide bond between glutamine and lysine, as the original reaction of the enzyme. When a deamidated protein is used in food, it is necessary to deamidate glutamine after protection of the xcex5-amino group with a reversible protecting group such as citraconyl group, to remove the protecting group thereafter and then to separate the deamidated protein from the released citraconic acid. It is evident that these steps sharply increase the production cost and are far from the realization.
In the case of the peptideglutaminase method, on the other hand, it is necessary to use a protein hydrolysate, because this enzyme hardly reacts upon protein but acts only upon a low molecular weight peptide and cannot therefore be applied to raw protein.
In consequence, though the reaction selectivity due to high substrate specificity of enzymes is originally one of the greatest advantages of the enzymatic method, which surpasses chemical and physical methods, it is the present situation that the enzymatic method cannot be put into practical use for the purpose of effecting deamidation of protein because of the absence of an enzyme which does not generate side reactions and is suited for the deamidation of high molecular weight protein.
Thus, though the deamidation of protein is an excellent modification method which will result in the great functional improvement, both of the conventional chemical and enzymatic methods have disadvantages, and their realization therefore is not completed yet.
In view of the above, the inventors of the present invention have conducted extensive studies on the screening of an inexpensive microorganism to be used as the source of an enzyme capable of directly acting upon amido groups which are bonded to protein and thereby effecting deamidation of the protein and, as a result, found that a new strain belonging to the genus Chryseobacterium, newly isolated from a soil sample by the present inventors, can produce an enzyme which exerts the deamidation function by directly acting upon amido groups in the bonded state in protein without accompanying cutting of peptid bonding and cross-linking of protein molecules. The present invention has been accomplished on the basis of this finding. In this specification, an enzyme which has the aforementioned actions is called a xe2x80x9cprotein-deamidating enzymexe2x80x9d.
Thereafter, the present inventors have selected strains belonging to the genus Chryseobacterium randomly from type cultures and examined their ability to produce the protein-deamidating enzyme. As the results, the productivity of this enzyme was found in all of the selected strains, as well as in other strains which belong, for example, to the genera Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium and Myroides. Each of these strains is a bacterium classified into Cytophagales or Actinomycetes, and the bacteria belong to the genus Chryseobacterium, Empedobacter, Flavobacterium, and Myroides are classified into Flavobacteriaceae.
The present inventors have then isolated and purified the protein-deamidating enzyme, determined nucleotide sequence of the gene coding for said protein-deamidating enzyme and confirmed that the protein-deamidating enzyme can be produced using a transformant transformed with a vector containing said gene.
Accordingly, the present invention relates to a method for the production of a protein-deamidating enzyme using a microorganism capable of producing the protein-deamidating enzyme, a method for the modification of protein using the protein-deamidating enzyme, a composition for use in the modification of protein, which comprises the protein-deamidating enzyme as the active ingredient, a method for the improvement of functionality of protein using the protein-deamidating enzyme, a method for the improvement of functionality of food using the protein-deamidating enzyme, a method for the improvement of extraction efficiency of protein and/or peptide using the protein-deamidating enzyme and a method for the control of transglutaminase reaction using the protein-deamidating enzyme.
The present invention also relates to a protein-deamidating enzyme, a gene which encodes said enzyme, a vector which contains said gene, a transformant transformed with said vector and a method for the production of the protein-deamidating enzyme, which comprises culturing said transformant in a medium, thereby allowing the transformant to produce the protein- deamidating enzyme, and subsequently collecting the protein-deamidating enzyme from the culture mixture, and the like.